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Open Access Research article

Leveraging two-way probe-level block design for identifying differential gene expression with high-density oligonucleotide arrays

Leah Barrera12, Chris Benner12, Yong-Chuan Tao3, Elizabeth Winzeler14 and Yingyao Zhou1*

Author Affiliations

1 Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, California 92121, USA

2 Bioinformatics Graduate Program, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA

3 Novartis Institutes for Biomedical Research, 100 Technology Square, Cambridge, MA 02139, USA

4 The Scripps Research Institute, La Jolla, California 92037, USA

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BMC Bioinformatics 2004, 5:42  doi:10.1186/1471-2105-5-42

Published: 20 April 2004



To identify differentially expressed genes across experimental conditions in oligonucleotide microarray experiments, existing statistical methods commonly use a summary of probe-level expression data for each probe set and compare replicates of these values across conditions using a form of the t-test or rank sum test. Here we propose the use of a statistical method that takes advantage of the built-in redundancy architecture of high-density oligonucleotide arrays.


We employ parametric and nonparametric variants of two-way analysis of variance (ANOVA) on probe-level data to account for probe-level variation, and use the false-discovery rate (FDR) to account for simultaneous testing on thousands of genes (multiple testing problem). Using publicly available data sets, we systematically compared the performance of parametric two-way ANOVA and the nonparametric Mack-Skillings test to the t-test and Wilcoxon rank-sum test for detecting differentially expressed genes at varying levels of fold change, concentration, and sample size. Using receiver operating characteristic (ROC) curve comparisons, we observed that two-way methods with FDR control on sample sizes with 2–3 replicates exhibits the same high sensitivity and specificity as a t-test with FDR control on sample sizes with 6–9 replicates in detecting at least two-fold change.


Our results suggest that the two-way ANOVA methods using probe-level data are substantially more powerful tests for detecting differential gene expression than corresponding methods for probe-set level data.

gene expression analysis; differential expression; high-density oligonucleotide array; ANOVA; FDR